In mammalian cells, peptides are transported in and out of cells by several different transport carriers. Functionally, there are transporters responsible for the influx of peptides into the cell and transporters responsible for the efflux of peptides out of the cells. Influx transporters transport small peptides and related compounds into the cytoplasm, and are indirectly linked to an energy source through ion gradients. Efflux transporters consist of several different transporters that function to remove peptides from the cytoplasm. These include the P-glycoprotein that removes a number of oncolytics as well as hydrophobic peptides (Endicott and Ling, 1989, Annu. Rev. Biochem. 58:137-171; Sharma et al. 1992, J. Biol. Chem. 267:5731-5734).
The present invention relates to peptide transporters responsible for influx of peptides into cells or organelles. This class of peptide transporters is located in the gastrointestinal tract, kidney, placenta, and liver lysosomes (Ganapathy et al., 1991, Indian J. Biochem. Biophys. 28:317-323; Skopicki et al., 1991, Am. J. Physiol. 261:F670-F678; Ganapathy et al., 1981, J. Biol. Chem. 256:118-124; Bird and Lloyd, 1990, Biochim. Biophys. Acta 1024:267-270).
Generally, the influx peptide transporter is located in the brush border of the epithelial cells of the mucosa. Properties of the transporter have been studied in situ in intestinal mucosa preparations and in vitro with brush border membrane vesicles, isolated enterocytes, and cell culture. Studies have been conducted with preparations from the rat, hamster, rabbit, chicken, Japanese newt, and humans (Ganapathy and Leibach, 1991, Curr. Biol. 3:695-701; Said et al., 1988, Biochim. Biophys. Acta 941: 232-240; Kramer et al., 1988, Biochim. Biophys. Acta 939: 167-172; Colonge et al., 1990, Am. J. Physiol. 259:G775-G780; Shimada and Hoshi, 1986, Jpn. J. Physiol 36: 451-465; Matthews and Burston, 1984, Clinical Sci., 67:541-549). Many different solutes including small peptides (di- and tripeptides), antibiotics (including several oral .beta.-lactams), oral angiotensin converting enzyme (ACE) inhibitors, and oral renin inhibitors are transported into the cytoplasm of the enterocyte by the influx peptide transporter (Ganapathy and Leibach, 1991, Curr. Biol. 3:695-701; Okano et al., 1986, J. Biol. Chem. 261:14130-14134; Nakashima et al., 1984, Biochem. Pharm. 33:3345-3352; Muranushi et al., 1989, Pharm. Res. 6:308-312; Friedman and Amidon, 1989, Pharm. Res. 6:1043-1047; Friedman and Amidon, 1990, J. Control Rel. 13:141-146; Kramer, 1991, 17th International Congress of Chemotherapy, June 23-28, Berlin, F.R.G., Abstract No. 1415).
The influx peptide transporter plays a pivotal role in the absorption of certain oral drugs including .beta.-lactam and ACE inhibitors. Out of 27 .beta.-lactam antibiotics examined, the influx peptide transporter was able to distinguish between those that are orally absorbed in humans and those that are not (Tabas et al., 1991, 31st Interscience Conference on Antimicrobial Agents and Chemotherapy Abstract No. 164). Moreover, the influx peptide transporter has been demonstrated to transport a number of oral .beta.-lactam antibiotics but not parenteral .beta.-lactam antibiotics in studies using human intestinal Caco-2 cells and rabbit intestinal brush-border membranes (Dantzig et al., 1992, Biochim. Biophys, Acta 1112:167-173; Dantzig et al., 1992, 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, Calif., Abstract No. 1460; Snyder et al., 1992, 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy Abstract No. 1461; Okano et al., 1986, J. Biol. Chem. 261:14130-14134. ) Similar studies have been conducted to examine the ability of the influx peptide transporter to predict which ACE inhibitors are orally absorbed (Friedman and Amidon, 1989, Pharm. Res. 6:1043-1047).
The influx peptide transporter is sodium independent, energy dependent, cotransports protons with the substrate ("proton-dependent"), and exhibits the ability to concentrate the substrate to higher levels within the cell than is present outside the cell (Hoshi, 1986, Ion Gradient-Coupled Transport, INSERM symposium No. 26. Editors: F. Alvarado and C. H. van Os, Elsevier Science Publishers; Ganapathy and Leibach, 1991, Curr. Opinion Cell Biol. 3:695-701; Ganapathy et al., 1991, Indian J. Biochem. Biophys. 28:317-323). The substrate specificity of the influx peptide transporter has been examined in several species and appears to be quite similar if not identical (Inui et al., 1992, J. Pharmacol. Exp. Thera. 260:482-486; Ganapathy and Leibach, 1983, J. Biol. Chem. 258:14189-14192; Yasumoto and Sugiyama, 1980, Agric. Biol. Chem. 44:1339-1344; Nakashima et al., 1984, Biochem. Pharmacol. 33:3345-3352; Okano et al., 1986, Biochem. Pharmacol. 35:1781-1786). The binding site of the influx peptide transporter is not known and, consequently, absolute chemical structural features necessary for binding and transport of solutes are also unknown. Structural-activity relationship studies of substrates and inhibitors have been conducted to elucidate some of the structural features required for transport (Bai et al., 1991, Pharm. Res. 8:593-599; Snyder et al., 1992, 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Oct. 11-14, Anaheim, Calif., Abstract No. 1461).
Influx peptide transporter activity has been identified as a 127,000 dalton membrane protein from rabbit intestinal mucosa by photoaffinity labeling methods employing radiolabeled penicillin or a radiolabeled cephalexin analog (Kramer, 1987, Biochim. Biophys. Acta 905:65-74; Kramer et al, 1988, Biochem Pharmacol. 37:247-2435). A purified 127,000 dalton protein from rabbit intestinal mucosa preparations reconstituted into liposomes resulted in binding and transport activities (Kramer et al., 1990, Biochim. Biophys. Acta 1030:50-59). The rabbit influx peptide transporter has been functionally expressed in Xenopus laevis oocytes (Miyamoto et al., 1991, J. Biol. Chem. 266:4742-4745). However, the structure of the cloned gene encoding the mammalian influx peptide transporter, or any component of it, has not been reported for any species.
Cloning of the influx peptide transporter would be useful for development of a method permitting the rapid identification and development of orally absorbed drugs that use this mechanism. Oral bioavailability is a highly desired property of many medications. Determination of the oral bioavailability of a drug at an early stage of development would be particularly advantageous. Presently, drugs are initially evaluated for oral bioavailability animal models. This process requires selection of only a few compounds whose synthesis must be scaled up to be evaluated in these models. If the compounds are not orally absorbed using these models, analogs of the compounds are often made in an effort to achieve oral bioavailability. This process is time consuming, laborious, and expensive. Further, there are many examples of Compounds that are well absorbed in animal models but not absorbed by humans. Other methods for evaluation are needed to complement this traditional approach.